粘红酵母利用纤维素水解液发酵制备微生物油脂的研究
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摘要
能源与环境问题是当今世界各国所面临的两大难题。伴随着日趋严重的全球性能源短缺与环境恶化,从环境保护与资源开发的角度出发,积极开发替代化石燃料的可再生新能源是一种必然趋势,如生物柴油就是一种具有很大发展潜力的可再生清洁能源。目前,生物柴油原料油脂的主要来源是动植物脂肪以及餐饮废油,存在成本高以及收集和运输困难的问题,因此寻找一种廉价的原料成为了生物柴油产业化的关键。某些微生物能将五碳糖和六碳糖转化为油脂,这一特性适用于对木质纤维素水解糖化液的利用。因此微生物油脂可为生物柴油的制备提供更加廉价而广泛的原料。
本文首先考察了粘红酵母As2.107在摇床及1L发酵罐上的发酵特性。结果表明:葡萄糖浓度在100g/L以下范围内不会对菌体造成抑制。有机氮为氮源时生物量高于以无机氮为氮源,但其油脂产量低于后者。以KNO3为氮源能够显著促进细胞的生长和油脂的积累。最佳的钾离子浓度为4g/L。在1L发酵罐上粘红酵母细胞生物量可达17.7g/L,油脂产量可达6.4g/L,最优化的培养条件为pH为6,温度为30℃,装液量为700mL,通气量为1vvm,转速为550 rpm。
研究了补料培养对粘红酵母细胞生长及油脂积累的影响,补糖有利于油脂产量的提高。最佳的补料方式为:发酵48小时一次补加14g/L的葡萄糖。采用该补料策略,细胞生物量可达16.8g/L,油脂产量可达9.1g/L。
研究了粘红酵母以工业木糖及混合糖为碳源发酵生产微生物油脂特性。结果表明,高的初始木糖浓度对粘红酵母细胞生长有一定的抑制作用,确定初始木糖浓度为35g/L,硝酸钾和酵母粉作为混合氮源,酵母粉浓度为4g/L,碳氮比为55,接种量为15%。H2PO4-的浓度为6g/L。
当以混合糖为碳源,酵母粉最佳浓度为4g/L,碳氮比为55,接种量为10%,萄糖抑制粘红酵母对木糖的代谢,最佳的木糖和葡萄糖比例为2.5:1。当木糖和葡萄糖的比例按照2.5:1,补料时间为发酵72~84小时,补料浓度为15~25g/L时,细胞生物量、油脂产量和油脂含量可分别达到19.5g/L、11.6g/L和59.5%。
本文还考察了木质纤维素水解液中糠醛和乙酸对菌体生长的影响。当糠醛和乙酸浓度大于2g/L时菌体完全不生长,糠醛和乙酸浓度为1g/L以下,随着糠醛浓度的降低,菌体生物量呈上升趋势,此时,糠醛对菌体生长的抑制作用大于乙酸。采用减压蒸馏脱出糠醛,并以浓缩的蔗渣和玉米芯水解液为碳源发酵制备微生物油脂,其生物量分别达到15.1g/L和13.7g/L,油脂含量分别达到32.5%和29.2%。因此,当甘蔗渣和玉米芯等纤维素原料水解并脱除糠醛和乙酸后,可以作为粘红酵母As2.107发酵制备油脂的原料。
将微生物油脂作为原料,采用超临界反应制备生物柴油,对产物成分进行气相色谱分析表明微生物油脂可作为制备生物柴油的原料。
At present, the world are facing with the serious energy and enviromental problem .With the problem of global energy shortage and environmental deterioration become more and more seriously, people are exploring the renewable energy to substitute the fossil fuel. Biodesel is such a kind of clean and renewable energy with great development potential. The raw materials of biodiesel are vegetable oil and waste oil at present . However, the high material price of vegetable oil and waste oil led to the high production cost of biodiesel production. So develop cheep materials are very important for the biodiesel industrialization. Some studies find that many microbe have a great deal of lipid in the body and can co-fermentate glucose and xylose simultaneously, so production of lipid material by microbial fermentation using hemicellulosic hydrolysate can provide a wide range and cheap raw materials for biodiesel.
First of all ,the batch and feed-batch fermentation in shaker and fermentor by fermentation of glucose with rhodotorula gutini As2.107 was investigated respectively.The experimental results showed that: the yeast growth will not be inhibited by glucose when the sugar concentration under 100 g/L, and the optimum initial glucose concentration is about 50g/L, the biomass of using organic nitrogen is higer than inorganic nitrogen but the lipid content of later is higher than the former, because of offering a higher kalium and lower natrium for the cell, using potassium nitrate under 4g/L as nitrogen source will promote lipids accumulation, the biomass and lipid can reach 17.7g/L and 6.4g/L while growing in 1L fermentor in which the optium operation is that ventilation was 1vvm , pH is 6 and stirrer speed was 550 rpm.
The feed-batch fermentation in shaker by fermentation of glucose with rhodotorula gutini As2.107 was investigated. The biomass and lipid can reach 16.8g/L and 9.1g/L while adding glucose(14 g/L) in the medium at 48h during fermentation.
The batch fermentation in shaker by fermentation of xylose with rhodotorula gutini As2.107 was investigated. The effects of fermentation operation parameters on fermentation process and lipid synthesize were discussed. The results showed that: the yeast growth will be inhibited by the high concentration of xylose, both of the biomass and content of lipid were maximum when xylose concentration is about 35g/L, KNO3 and yeast were used as nitrogen source, the yeast concentration is about 4g/L ,the ratio of carbon and nitrogen was 55, inoculation amount was 15% and the concentration of KH2PO4 is 6g/L.
The batch fermentation in shaker by cofermentation of glucose and xylose with rhodotorula gutini As2.107 was investigated. The results showed that: both of the biomass and content of lipid were maximum when the ratio of xylose and glucose is 2.5, the total concentration of sugar is about 50g/L, KNO3 and yeast were used as a nitrogen source, the yeast concentration is about 4g/L ,the ratio of carbon and nitrogen was 55, When grown on mixed sugar containing glucose (15g/L) and xylose (35g/L) under feed-batch fermentation which adding 15~25g/L sugar (ratio of xylose and glucose is 2.5) 72~84h after beginning fermentation, biomass and lipid content were19.5 g/L and 59.5% respectively.
The influence of furfural and acetic acid on Rhodotorula glutin As2.107 growth was investigated. The yeast will stop growth when their concentrations exceeded 2 g/L. When grown on bagasse and corncob, biomass were15.1g/L and 13.7g/L,lipid content were 32.5% and 29.2%. So the hemicellulosic hydrolysate can be used as raw material to produce microbial lipid when removal of toxin from it. The lipid produced by fermentation which using lignocellulosic hydrolysate as raw material is also can be used to prepare of biodiesel.
本文首先考察了粘红酵母As2.107在摇床及1L发酵罐上的发酵特性。结果表明:葡萄糖浓度在100g/L以下范围内不会对菌体造成抑制。有机氮为氮源时生物量高于以无机氮为氮源,但其油脂产量低于后者。以KNO3为氮源能够显著促进细胞的生长和油脂的积累。最佳的钾离子浓度为4g/L。在1L发酵罐上粘红酵母细胞生物量可达17.7g/L,油脂产量可达6.4g/L,最优化的培养条件为pH为6,温度为30℃,装液量为700mL,通气量为1vvm,转速为550 rpm。
研究了补料培养对粘红酵母细胞生长及油脂积累的影响,补糖有利于油脂产量的提高。最佳的补料方式为:发酵48小时一次补加14g/L的葡萄糖。采用该补料策略,细胞生物量可达16.8g/L,油脂产量可达9.1g/L。
研究了粘红酵母以工业木糖及混合糖为碳源发酵生产微生物油脂特性。结果表明,高的初始木糖浓度对粘红酵母细胞生长有一定的抑制作用,确定初始木糖浓度为35g/L,硝酸钾和酵母粉作为混合氮源,酵母粉浓度为4g/L,碳氮比为55,接种量为15%。H2PO4-的浓度为6g/L。
当以混合糖为碳源,酵母粉最佳浓度为4g/L,碳氮比为55,接种量为10%,萄糖抑制粘红酵母对木糖的代谢,最佳的木糖和葡萄糖比例为2.5:1。当木糖和葡萄糖的比例按照2.5:1,补料时间为发酵72~84小时,补料浓度为15~25g/L时,细胞生物量、油脂产量和油脂含量可分别达到19.5g/L、11.6g/L和59.5%。
本文还考察了木质纤维素水解液中糠醛和乙酸对菌体生长的影响。当糠醛和乙酸浓度大于2g/L时菌体完全不生长,糠醛和乙酸浓度为1g/L以下,随着糠醛浓度的降低,菌体生物量呈上升趋势,此时,糠醛对菌体生长的抑制作用大于乙酸。采用减压蒸馏脱出糠醛,并以浓缩的蔗渣和玉米芯水解液为碳源发酵制备微生物油脂,其生物量分别达到15.1g/L和13.7g/L,油脂含量分别达到32.5%和29.2%。因此,当甘蔗渣和玉米芯等纤维素原料水解并脱除糠醛和乙酸后,可以作为粘红酵母As2.107发酵制备油脂的原料。
将微生物油脂作为原料,采用超临界反应制备生物柴油,对产物成分进行气相色谱分析表明微生物油脂可作为制备生物柴油的原料。
At present, the world are facing with the serious energy and enviromental problem .With the problem of global energy shortage and environmental deterioration become more and more seriously, people are exploring the renewable energy to substitute the fossil fuel. Biodesel is such a kind of clean and renewable energy with great development potential. The raw materials of biodiesel are vegetable oil and waste oil at present . However, the high material price of vegetable oil and waste oil led to the high production cost of biodiesel production. So develop cheep materials are very important for the biodiesel industrialization. Some studies find that many microbe have a great deal of lipid in the body and can co-fermentate glucose and xylose simultaneously, so production of lipid material by microbial fermentation using hemicellulosic hydrolysate can provide a wide range and cheap raw materials for biodiesel.
First of all ,the batch and feed-batch fermentation in shaker and fermentor by fermentation of glucose with rhodotorula gutini As2.107 was investigated respectively.The experimental results showed that: the yeast growth will not be inhibited by glucose when the sugar concentration under 100 g/L, and the optimum initial glucose concentration is about 50g/L, the biomass of using organic nitrogen is higer than inorganic nitrogen but the lipid content of later is higher than the former, because of offering a higher kalium and lower natrium for the cell, using potassium nitrate under 4g/L as nitrogen source will promote lipids accumulation, the biomass and lipid can reach 17.7g/L and 6.4g/L while growing in 1L fermentor in which the optium operation is that ventilation was 1vvm , pH is 6 and stirrer speed was 550 rpm.
The feed-batch fermentation in shaker by fermentation of glucose with rhodotorula gutini As2.107 was investigated. The biomass and lipid can reach 16.8g/L and 9.1g/L while adding glucose(14 g/L) in the medium at 48h during fermentation.
The batch fermentation in shaker by fermentation of xylose with rhodotorula gutini As2.107 was investigated. The effects of fermentation operation parameters on fermentation process and lipid synthesize were discussed. The results showed that: the yeast growth will be inhibited by the high concentration of xylose, both of the biomass and content of lipid were maximum when xylose concentration is about 35g/L, KNO3 and yeast were used as nitrogen source, the yeast concentration is about 4g/L ,the ratio of carbon and nitrogen was 55, inoculation amount was 15% and the concentration of KH2PO4 is 6g/L.
The batch fermentation in shaker by cofermentation of glucose and xylose with rhodotorula gutini As2.107 was investigated. The results showed that: both of the biomass and content of lipid were maximum when the ratio of xylose and glucose is 2.5, the total concentration of sugar is about 50g/L, KNO3 and yeast were used as a nitrogen source, the yeast concentration is about 4g/L ,the ratio of carbon and nitrogen was 55, When grown on mixed sugar containing glucose (15g/L) and xylose (35g/L) under feed-batch fermentation which adding 15~25g/L sugar (ratio of xylose and glucose is 2.5) 72~84h after beginning fermentation, biomass and lipid content were19.5 g/L and 59.5% respectively.
The influence of furfural and acetic acid on Rhodotorula glutin As2.107 growth was investigated. The yeast will stop growth when their concentrations exceeded 2 g/L. When grown on bagasse and corncob, biomass were15.1g/L and 13.7g/L,lipid content were 32.5% and 29.2%. So the hemicellulosic hydrolysate can be used as raw material to produce microbial lipid when removal of toxin from it. The lipid produced by fermentation which using lignocellulosic hydrolysate as raw material is also can be used to prepare of biodiesel.
引文
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